I .M. Sokolova, V.J. Berger J. Exp. Mar. Biol. Ecol. 245 2000 1 –23
3
the study Sokolova, 1997. Previous studies Sokolova et al., 1997 have suggested that the phenotypic differentiation along a salinity gradient cannot be explained by selective
pressures imposed by the differences in crypsis and or heating properties of the shell colour morphs involved and probably implies physiological selection.
In order to test the hypothesis about physiological selection as a driving force shaping pheno- geno-typic structure of L
. saxatilis populations along a salinity gradient in White Sea estuaries, we investigated physiological responses of different shell colour
morphs of L . saxatilis to salinity variation and also to the combination of low salinity
and subzero temperatures which may be expected in White Sea estuaries in winter. Particular attention was given to the phenotypes which are most common in the studied
populations of L . saxatilis and show the greatest and the most consistent difference in
abundance between the marine and estuarine sites purple tessellated unbanded and brown tessellated unbanded. Since it has been shown that the nature and the
mechanisms of salinity adaptations depend on the degree of environmental disturbance Kinne, 1964, 1971; Precht and Plett, 1979; Berger and Kharazova, 1997, we analysed
responses of different shell colour morphs of L
. saxatilis to moderate and extreme salinity changes. This allowed a comparison to assess the relationship between
genetically determined shell colour polymorphism and physiological variation with respect to salinity in more detail.
2. Materials and methods
2.1. Study sites Snails were collected in July–August, 1995 in Chupa, Keret and Kolvitsa Inlets of the
Kandalaksha Bay of the White Sea Fig. 1. In the estuaries, snails were collected close to the distribution limit of L
. saxatilis in the head of the estuaries in the Keret E-1 and the Kolvitsa E-2 Inlets Fig. 1. Measurements of the surface salinity in summer and
autumn showed that salinity at low tides was usually below 10‰ in these sites, sometimes dropping down to 1–3‰. Overall, salinity was below 15‰ at all phases of
the tidal cycle in these sites. In the Chupa Inlet, two populations from the marine sites were sampled M-1 and M-2 Fig. 1. Here surface salinity was relatively constant and
varied between 24 and 26‰. This is the normal salinity of the surface waters in the White Sea in summer and autumn Kuznetsov, 1960.
Most analyses were done in animals from sites M-1 and E-1. Animals from sites M-2 and E-2 were only used in one exp. 3 or two experiments exp. 2 and exp. 6,
respectively, due to a limited amount of snails available. 2.2. Experimental methods
Snails were transferred to the laboratory within a few hours populations M-1, M-2 and E-1 or 1 day E-2 after collection. Prior to the experiments, they were acclimated
for 2–5 days at 10618C and 24–25‰ in aquaria with well-aerated recirculating natural sea water. No food was allowed. The temperature was chosen to approximate the natural
4 I
.M. Sokolova, V.J. Berger J. Exp. Mar. Biol. Ecol. 245 2000 1 –23
Fig. 1. Study sites in the Kandalaksha Bay B of the White Sea A. Position of the two marine M-1 and M-2 and two estuarine E-1 and E-2 study sites is shown. Small boxes: C, Kolvitsa Inlet; D, Chupa and Keret
Inlets.
conditions at the collection sites. It has been previously shown that acclimation to the optimum salinity which is ca. 24–26‰ for the White Sea L
. saxatilis is very fast in intertidal gastropods and usually takes from several hours to a few days depending on
the length of the preceding history at suboptimal salinity Khlebovich and Berger, 1975. This suggests that an acclimation to laboratory conditions was fully accomplished in the
studied periwinkles prior to the start of the experiments.
In each trial, two to seven shell colour variants were involved Table 1. Shell colour was scored according to Sergievsky et al. 1995 using a combination of three traits —
shell ground colour C, presence absence of tessellations of the colour different from the ground one S and number of longitudinal bands B. Particular state of each trait
was designated in a subscript or a superscript. In some snails, shell colour pattern was not distinguishable due to the heavy calcification of the shell upper layer. Such shells
appear greyish-white W or pure white W . In each experiment, two commonest shell
2 3
p 1
colour morphs purple tessellated and brown tessellated unbanded, C S B and
b 1
C S B were included, which account for the major differences in phenotypic structure between the estuarine and marine populations of L
. saxatilis Sokolova, 1997; Sokolova et al., 1997. Other shell colour variants were occasionally included in the experiments,
when the amount of available animals was sufficient for the analysis. Sea water of different salinity was obtained by a dilution of natural sea water with
I .M. Sokolova, V.J. Berger J. Exp. Mar. Biol. Ecol. 245 2000 1 –23
5 Table 1
a
Shell colour morphs of L . saxatilis used in the experiments
Abbreviation Calcification
Ground Tessellations
Bands Relative abundance of the
colour morphs in populations:
M-1 M-2
E-1 E-2
p 1
C S B 2
Dark purple 1
67.3 75.5
53.7 52.8
p 2
C S B 2
Dark purple 2
8.2 11.2
4.9 NF
b 1
C S B 2
Brown 1
16.8 9.4
40.7 47.2
o 1
C S B 2
Orange 1
NF ,1.0
NF NF
W and W 1
? ?
NF 2.4
NF NF
2 3
W B 1
? ?
2 ,1.0
,1.0 NF
NF
3 2
a
Particular states of every trait are given in subscripts or superscripts, e.g. shell ground colour C: p, purple; b, brown, o, orange; tessellations S: 1, present; 2, absent; longitudinal bands B: 0, absent; 2,
two-banded morphs; degree of calcification W: 2, moderate; 3, heavy. Calcification of shell upper layer: 1, present; 2, absent. Presence absence of intensive calcification of the upper shell layer is presumably
determined by a separate locus in White Sea L . saxatilis Kozminsky et al., 1995. This calcification is
superimposed over other shell colour variants, therefore it is impossible to distinguish ground colour and tessellation pattern in the white morphs W , W and W B . Shell colour morphs W and W are intergradable,
2 3
3 2
2 3
though normally they can be distinguished by a shade of the white W is greyish-white, and W is pure white.
2 3
NF, shell colour variant was not found.
natural filtered fresh water from the nearby Krivoye Lake and controlled with a densitometer in order to keep deviation from the intended salinity value below 0.3‰.
In physiological experiments it is very important to standardise experimental animals as scrupulously as possible according to age, reproductive state, infection disease etc.
Therefore, in most experiments only adult sexually mature snails 5–8 mm shell diameter were used. The only experiment in which a second age size group juveniles
of 3–4 mm shell diameter, was experiment 6 as juveniles were not available in amounts sufficient for all analyses. To check for the trematode infection, dissection of the
experimental animals is required. However, due to the time limitations this was not done. Instead, the pilot comparisons were performed in order: 1 to check for the
differences in the tested physiological responses of infected and uninfected L
. saxatilis irrespective of the shell colour, and 2 to compare infection prevalence in snails with
different shell colour in the studied populations. It was found that trematode infection did not significantly influence oxygen consumption, rate of salt loss, mortality in fresh
water or freeze resistance of L . saxatilis Sokolova, unpublished data. This is in
agreement with the results of previous studies which showed no differences in oxygen consumption, resistance to fresh water, and freezing tolerance between infected and
uninfected Littorina spp. Lyzen et al., 1992; Galaktionov, 1993; Sokolova, 1997. Furthermore, no differences in the trematode infection levels were detected between the
2
studied shell colour morphs in either studied population x 5 0.09–0.36, df 5 1,
P 5 0.45–0.77. Thus, the periwinkles were used in further experiments without special check for the trematode infection.
2.2.1. Experiment 1: oxygen consumption in low salinity Oxygen consumption rate was determined in snails from the estuarine population E-1
and the marine site M-1 at 108C in salinity of the preliminary acclimation 24‰,
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.M. Sokolova, V.J. Berger J. Exp. Mar. Biol. Ecol. 245 2000 1 –23
controls and after different exposure periods to low salinity 14‰. The latter value was chosen because it was the lower salinity limit at which all snails were able to maintain
activity and did not withdraw into the shell. The same individuals were used to measure oxygen consumption in the control and after different exposure periods 1 day and 13
days in the population M-1 and 1 day and 7.5 days in the population E-1. Ten to nine
p 1
b 1
animals for each of the two main shell colour morphs C S B and C S B were used. Oxygen consumption was measured using the method of closed respiration chambers.
Snails were placed individually in the air-tight bottles 50–60 ml half-filled with the air-saturated sea water of respective salinity at 108C and left for 30–60 min to reduce
effect of handling. After this, water was carefully drained off using a plastic tubing in order to reduce disturbance to the snails, and the bottles were refilled. In each
experimental set, 2–3 empty bottles ‘blanks’ were filled with the water of the same salinity and temperature. Bottles were sealed with air-tight corks and left for 8–10 h at
108C. Decrease in the oxygen concentration after this exposure period never exceeded 25–30 of the starting value. After the exposure, concentration of oxygen in the water
was analytically determined by the Winkler method Golterman, 1983. Snails were removed from the bottles, blotted dry and weighted to the nearest 1 mg. Wet weight of
the snails ranged between 110 and 240 mg population E-1, and 60 and 160 mg population M-1.
21 21
~ Oxygen consumption rate MO was calculated as mg O h
g fresh weight. In
2 2
~ the only case when MO
was found to depend significantly on the weight of
2
experimental animals in the snails from the population M-1 after 13 days of exposure in 14‰, respiration rates were standardised to the average weight in the experimental
group according to the formula 1:
b
W
i
] R 5 R 3
1
S D
st i
¯ W
21 21
where R is a standardised respiration rate mg O h g
, R is the respiration rate of
st 2
i 21
21
¯ ith animal mg O h
g , W is the fresh live weight of ith animal g, W is the
2 i
average fresh weight in the experimental group g, and b is a power coefficient in the
b
equation: R 5 aW . This coefficient was obtained by a calculation of the linear regression relating log W to log R by the least square method Sokal and Rohlf, 1995.
~ In final calculations, MO was expressed in relative units percent from the control level
2
in the same individual. For the control animals, respiration rates were expressed as percent of the respective mean control values.
2.2.2. Experiment 2: activity in low salinity Snails from the populations E-1, E-2 and M-1 were used in the experiment. Animals
were placed into 1–2 large Petri dishes with the water of 8 or 10‰ at 108C and covered with a glass lid. Salinity values of 8 and 10‰ were chosen in order: 1 to imitate
brackish water conditions in the estuarine habitats of White Sea L . saxatilis and 2 to
evoke differentiated response in the studied periwinkles, as only some individuals were able to maintain activity under these conditions while others ceased activity and isolated
themselves inside the shells. At salinities higher than 10‰ or lower than 8‰, 100 or 0 of active animals, respectively, were observed at least in some of the studied
I .M. Sokolova, V.J. Berger J. Exp. Mar. Biol. Ecol. 245 2000 1 –23
7
populations. After 1, 2, 4 and 6 h of exposure, active and non-active withdrawn into the shell and isolated by the operculum periwinkles were counted. At each scoring period,
water in the Petri dishes was changed. Care was taken not to disturb snails during this
p 1
b 1
procedure. We used 50–60 and 25–60 animals of C S B and C S B
morphs, respectively.
2.2.3. Experiment 3: rates of isolating and opening responses Rates of the behavioural response to fast and abrupt salinity change were tested in the
animals from the populations M-1, M-2 and E-1. Experiments were carried out at 17–198C. Snails were placed individually into small vials 10 ml, fixed with the
aperture upwards and covered with the natural sea water for 10–15 min. Then the water was carefully removed. Under these conditions, snails protrude the foot trying to recover
the normal position. When the foot was fully extended, the vial was quickly filled with fresh water and time to close from the first contact with fresh water to the closure of the
operculum was measured to the nearest 1 s. After the measurements, snails were left for 45 min in fresh water, then fresh water was carefully removed and the vial quickly filled
with natural sea water. Time to open from the first contact with sea water to the emergence of tips of the tentacles was measured to the nearest 0.2 s. Different time
scales were used for measuring opening and closing times because it normally took much longer in the periwinkles to close than to open the shell. N was 6–12 for each
shell colour morph.
2.2.4. Experiment 4: rate of salt loss in fresh water Snails from the populations E-1 and M-1 were rinsed in plenty of deionised water to
remove salts from the shell surfaces and placed individually in 100-ml glass vials. Each vial was filled with 50 ml of NaCl solution in deionised water 3.5–4.0 mg l at
118–208C. The snails were left in the NaCl solution for 5 min, which was then well mixed. Its conductivity was measured using the reochord bridge according to the method
described in Khlebovich and Berger 1965. After 2 h of exposure, conductivity of the solution was measured again.
To transform conductivity into concentration units, calibration solutions of NaCl were used. Relationship between conductivity and salt concentration closely followed the
2
power function R 597–99.9. Power regression equations were used to calculate salt concentration in the solution at the start and after 2 h exposure. After the experiment,
snails were taken out of the vials, blotted dry and weighed to the nearest 1 mg. Rate of
21 21
salt loss was expressed as mg NaCl h g
live weight. N was 8–12 for each shell colour morph.
2.2.5. Experiment 5: mortality in fresh water Animals from the populations M-1 and E-1 were placed into the trays filled with
700–800 ml of fresh water at 108C. After specified exposure periods 7, 12 and 19 days, a portion of snails was removed from the tray, placed in sea water and allowed to
recover for 2 h at 108C. The longest exposure period was chosen to produce ,80 of mortality and was 12 days for snails from the population M-1 and 19 days for
individuals from the site E-1. After the recovery period, the number of dead and alive
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.M. Sokolova, V.J. Berger J. Exp. Mar. Biol. Ecol. 245 2000 1 –23
snails were counted. Those snails which failed to respond by contracture to being poked by the needle were considered dead. Mortality was expressed as percent of dead
individuals. N was 458–543 for the purple tessellated unbanded morph and 92–98 for the brown tessellated unbanded morph.
2.2.6. Experiment 6: resistance to low salinity and subzero temperatures Snails from the population E-2 were used to determine resistance to freezing at
29.08C and 8‰. This combination of temperature and salinity was chosen to approximate extreme conditions in White Sea estuaries which may be experienced by the
intertidal snails during low tides in late autumn. During this period, the ice cover over the intertidal which in winter shields the substrate against freezing, is not yet formed, but
frosty days with air temperature down to 29–108C are frequent Savoskin, 1967.
Animals were placed in small plastic cylinders, covered with 15–20 ml of diluted sea water and slowly cooled down and frozen at the rate of 1.5 h at 3–48C, 1.5 h at 218C
and then 2 h at 298C temperature range 29.0–9.58C. Temperature control showed that it took 90 min to reduce the temperature from 218C down to 298C, so the actual
exposure time at 298C was 30 min. After the exposure snails were slowly thawed at 3–48C for 3 h and at 15–178C for 2 h until ice crystals totally disappeared from the
water, placed into natural sea water 24–25‰ at 15–178C and allowed to recover for 14 h. This period was found to be sufficient for all alive snails to resume activity. After the
recovery period the number of dead snails was determined. N was 30 adults or 80 juveniles for each shell colour morph.
2.3. Statistics Rates of oxygen consumption, salt loss and rate of opening and closing of the aperture
were compared using standard ANOVA procedures Sokal and Rohlf, 1995. Prior to the analysis, data were checked for the fit to normal distribution and for the heterogeneity of
variances by chi-square or Kolmogorov–Smirnov tests and Cochran test, respectively. Assumptions of the normal distribution and homogeneity of variances was violated in
neither of the studied parameters except the time to close the aperture upon transfer to fresh water Table 3. For this variable, a log-transformation was used which resulted in
a significant improvement of homogeneity of the variances.
To compare mortality survivability and relative activity estimates, log-linear analysis followed by chi-square test or exact Fisher test if the expected frequencies were less
than 5 for pairwise comparisons were used Sokal and Rohlf, 1995. To choose the best-fitted model in log-linear analysis, an iterative procedure was performed Sokal and
Rohlf, 1995. Model was considered to fit the data if the probability level for the model PModel exceeded 0.10. An effect of factor interactions was included if the
improvement in the model fit i.e. an increase in G-values of goodness-of-fit was significant at the 5 level.
Results are expressed as percentages or means6standard errors if not mentioned otherwise.
I .M. Sokolova, V.J. Berger J. Exp. Mar. Biol. Ecol. 245 2000 1 –23
9
3. Results
